title: Generation of high-producing cell lines by overexpression of cell division cycle 25 homolog A in Chinese hamster ovary cells.
abstract: To improve the efficiency of conventional gene amplification systems, the effect of cell cycle modification during the gene amplification process on IgG production was investigated in Chinese hamster ovary (CHO) cells. The full-length cDNA of CHO cell division cycle 25 homolog A (Cdc25A) was introduced into CHO DG44 cells and the effects of CDC25A overexpression on the cell cycle, transgene copy number and IgG productivity were examined. Both wild-type and mutated CDC25A-overexpressing CHO cells showed a rapid increase in transgene copy number compared with mock cells during the gene amplification process, in both cell pools and individual clones. High-producing clones were obtained with high frequency in CDC25A-overexpressing cell pools. The specific production rate of the isolated clone CHO SD-S23 was up to 2.9-fold higher than that of mock cells in the presence of 250 nM methotrexate (MTX). Cell cycle analysis revealed that the G2 to M phase transition rate was increased ∼1.5-fold in CDC25A-overexpressing CHO cells under MTX treatment. Our results show the improvement of conventional gene amplification systems via cell cycle engineering at an early stage of cell line development.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Kyoung Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Tomomi
authors.initials: T
authors.lastname: Tsutsui
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Honda
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Asano
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
authors.forename: Hisao
authors.initials: H
authors.lastname: Ohtake
authors.forename: Takeshi
authors.initials: T
authors.lastname: Omasa
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002453:Cell Cycle / Q000235:genetics*; D002455:Cell Division / Q000235:genetics; D002999:Clone Cells; D006224:Cricetinae; D003412:Cricetulus; D005784:Gene Amplification; D018628:Gene Dosage; D005818:Genetic Engineering / Q000379:methods*; D008727:Methotrexate / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019076:Transgenes; D020687:cdc25 Phosphatases / Q000235:genetics* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D020687:cdc25 Phosphatases; D008727:Methotrexate
keywords: CDC25A; Cell cycle; Chinese hamster ovary cells; Gene amplification; Monoclonal antibody